Using flowViz to visualize flow cytometry data

Summary: Automated analysis of flow cytometry (FCM) data isessential for it to become successful as a high throughput technology.We believe that the principles of Trellis graphics can be adaptedto provide useful visualizations that can aid such automation.In this article, we describe the R/Bioconductor package flowVizthat implements such visualizations. Availability: flowViz is available as an R package from theBioconductor project: http://bioconductor.org Contact: dsarkar{at}fhcrc.org Associate Editor: Olga Troyanskaya     

Using quantum dots to visualize clathrin associations

Our current understanding of clathrin-mediated endocytosis proposes that the process is initiated at a specialized anatomical structure called a coated pit. Electron microscopy has been required for elucidation of the morphology of coated pits and the vesicles produced therein, and the presence of a bristle coat has been taken as suggestive of clathrin surrounding these vesicles. More recently, immunocytochemical methods have confirmed that endocytic vesicles are surrounded by clathrin and its adaptor proteins, but there is a need to identify precisely and to follow the fate of the cellular organelles seen by fluorescence microscopy. We used quantum immune-electron microscopy to localize clathrin in a human adrenal cortical cell line (SW-13). Clathrin was shown to associate with a variety of vesicle types including the classic clathrin-coated vesicles and pits used in receptor internalization, pentilaminar annular gap junction vesicles, and multivesicular bodies. The images obtained with quantum dot technology allow accurate and specific localization of clathrin and the clathrin adaptor protein, AP-2, with cellular organelles and suggest that some of the structures classified as typical coated vesicles by immunocytochemical light microscopic techniques actually may be membrane bound pits.     

Using consensus networks to visualize contradictory evidence for species phylogeny

Building species phylogenies from genome data requires the evaluation of phylogenetic evidence from independent gene loci. We propose an approach to do this using consensus networks. We compare gene trees for eight yeast genomes and show that consensus networks have potential for helping to visualize contradictory evidence for species phylogenies.     

Metal-induced DNA translocation leads to DNA polymerase conformational activation

Binding of the catalytic divalent ion to the ternary DNA polymerase β/gapped DNA/dNTP complex is thought to represent the final step in the assembly of the catalytic complex and is consequently a critical determinant of replicative fidelity. We have analyzed the effects of Mg(2+) and Zn(2+) on the conformational activation process based on NMR measurements of [methyl-(13)C]methionine DNA polymerase β. Unexpectedly, both divalent metals were able to produce a template base-dependent conformational activation of the polymerase/1-nt gapped DNA complex in the absence of a complementary incoming nucleotide, albeit with different temperature thresholds. This conformational activation is abolished by substituting Glu295 with lysine, thereby interrupting key hydrogen bonds necessary to stabilize the closed conformation. These and other results indicate that metal-binding can promote: translocation of the primer terminus base pair into the active site; expulsion of an unpaired pyrimidine, but not purine, base from the template-binding pocket; and motions of polymerase subdomains that close the active site. We also have performed pyrophosphorolysis studies that are consistent with predictions based on these results. These findings provide new insight into the relationships between conformational activation, enzyme activity and polymerase fidelity.     

Using networks to analyze and visualize the distribution of overlapping genes in virus genomes

Gene overlap occurs when two or more genes are encoded by the same nucleotides. This phenomenon is found in all taxonomic domains, but is particularly common in viruses, where it may increase the information content of compact genomes or influence the creation of new genes. Here we report a global comparative study of overlapping open reading frames (OvRFs) of 12,609 virus reference genomes in the NCBI database. We retrieved metadata associated with all annotated open reading frames (ORFs) in each genome record to calculate the number, length, and frameshift of OvRFs. Our results show that while the number of OvRFs increases with genome length, they tend to be shorter in longer genomes. The majority of overlaps involve +2 frameshifts, predominantly found in dsDNA viruses. Antisense overlaps in which one of the ORFs was encoded in the same frame on the opposite strand (−0) tend to be longer. Next, we develop a new graph-based representation of the distribution of overlaps among the ORFs of genomes in a given virus family. In the absence of an unambiguous partition of ORFs by homology at this taxonomic level, we used an alignment-free k-mer based approach to cluster protein coding sequences by similarity. We connect these clusters with two types of directed edges to indicate (1) that constituent ORFs are adjacent in one or more genomes, and (2) that these ORFs overlap. These adjacency graphs not only provide a natural visualization scheme, but also a novel statistical framework for analyzing the effects of gene- and genome-level attributes on the frequencies of overlaps.     

Avidity regulation of integrins: the driving force in leukocyte adhesion

The activity of integrins on leukocytes is tightly controlled, and their adhesion capacity shifts rapidly when cells emigrate from the blood to the tissues. The leukocyte-specific beta2 integrin LFA-1 (alphaLbeta2) is the most important integrin expressed by leukocytes that regulate lymphocyte migration and the initiation of an immune response through binding to ICAM-1,-2 or-3. The binding activity of LFA-1 is rapidly altered by intracellular stimuli that activate LFA-1. Although alterations in the affinity of LFA-1, which leads to enhanced ICAM-1 binding, have been proposed, evidence is emerging that dynamic reorganisation of LFA-1 into microclusters is the major mechanism that regulates its binding capacity.     

A mechanism for modulation of cellular responses to VEGF: activation of the integrins

Many similarities exist in the cellular responses elicited by VEGF and governed by integrins. Here, we identify a basis for these interrelationships: VEGF activates integrins. VEGF enhanced cell adhesion, migration, soluble ligand binding, and adenovirus gene transfer mediated by alphavbeta3 and also activated other integrins, alphavbeta5, alpha5beta1, and alpha2beta1, involved in angiogenesis. Certain tumor cells exhibited high spontaneous adhesion and migration, which were attributable to a VEGF-dependent autocrine/paracrine activation of integrins. This activation was mediated by the VEGFR2 receptor and regulated via phosphatidylinositol-3-kinase, Akt, and the PTEN signaling axis. Thus, integrin activation provides a mechanism for VEGF to induce a broad spectrum of cellular responses.     

The Rho GEFs LARG and GEF-H1 regulate the mechanical response to force on integrins

How individual cells respond to mechanical forces is of considerable interest to biologists as force affects many aspects of cell behaviour. The application of force on integrins triggers cytoskeletal rearrangements and growth of the associated adhesion complex, resulting in increased cellular stiffness, also known as reinforcement. Although RhoA has been shown to play a role during reinforcement, the molecular mechanisms that regulate its activity are unknown. By combining biochemical and biophysical approaches, we identified two guanine nucleotide exchange factors (GEFs), LARG and GEF-H1, as key molecules that regulate the cellular adaptation to force. We show that stimulation of integrins with tensional force triggers activation of these two GEFs and their recruitment to adhesion complexes. Surprisingly, activation of LARG and GEF-H1 involves distinct signalling pathways. Our results reveal that LARG is activated by the Src family tyrosine kinase Fyn, whereas GEF-H1 catalytic activity is enhanced by ERK downstream of a signalling cascade that includes FAK and Ras.     

S100A9 mediates neutrophil adhesion to fibronectin through activation of beta2 integrins

Neutrophil migration from the blood to inflammatory sites follows a cascade of events, in which adhesion to endothelial cells and extracellular matrix proteins is essential. S100A8, S100A9, and S100A12 are small abundant proteins found in human neutrophil cytosol and presumed to be involved in leukocyte migration. Here we investigated the S100 proteins' activities in neutrophil tissue migration by evaluating their effects on neutrophil adhesion to certain extracellular matrix proteins. S100A9 induced adhesion only to fibronectin and was the only S100 protein that stimulated neutrophil adhesion to this extracellular matrix protein. Experiments with blocking antibodies revealed that neither beta1 nor beta3 integrins were strongly involved in neutrophil adhesion to fibronectin, contrary to what the literature predicted. In contrast, neutrophil adhesion to fibronectin was completely inhibited by anti-beta2 integrins, suggesting that S100A9-induced specific activation of beta2 integrin is essential to neutrophil adhesion.     

Immunodiagnosis of platelet activation in immune thrombocytopenia through scFv antibodies cognate to activated IIb3 integrins

Immune thrombocytopenia (ITP) is an autoimmune bleeding disorder characterized by low platelet count and presence of IgG autoantibodies to platelet surface glycoproteins, such as $$\alpha$$IIbβ3 and GPIb/IX. Our previous work has shown that platelets in ITP patients exist in an activated state. Two different marker-based approaches are used to study the course of platelet activation: (1) binding of PAC-1 antibody, signifying a change in αIIbβ3 conformation, and (2) expression of P-selectin, signifying alpha granule content release from platelets. Here, we describe the development of a new scFv antibody (R38) that, compared with PAC-1, appears to better distinguish between platelets of ITP patients and healthy controls. Notably, R38 was generated using commercially sourced resting-state integrin that was coated on a microtiter plate. Its ability to distinguish between ITP patients and healthy controls thus suggests that inadvertent integrin activation caused by coating involves a conformational change and exposure of a cryptic epitope. This report also describes for the first time the potential use of an scFv antibody in the immunodiagnosis of platelet activation in ITP patients.     

Immunodiagnosis of platelet activation in immune thrombocytopenia through scFv antibodies cognate to activated IIb3 integrins

Immune thrombocytopenia (ITP) is an autoimmune bleeding disorder characterized by low platelet count and presence of IgG autoantibodies to platelet surface glycoproteins, such as αIIbβ3 and GPIb/IX. Our previous work has shown that platelets in ITP patients exist in an activated state. Two different marker-based approaches are used to study the course of platelet activation: (1) binding of PAC-1 antibody, signifying a change in αIIbβ3 conformation, and (2) expression of P-selectin, signifying alpha granule content release from platelets. Here, we describe the development of a new scFv antibody (R38) that, compared with PAC-1, appears to better distinguish between platelets of ITP patients and healthy controls. Notably, R38 was generated using commercially sourced resting-state integrin that was coated on a microtiter plate. Its ability to distinguish between ITP patients and healthy controls thus suggests that inadvertent integrin activation caused by coating involves a conformational change and exposure of a cryptic epitope. This report also describes for the first time the potential use of an scFv antibody in the immunodiagnosis of platelet activation in ITP patients.     

Resolution of conformational activation in the kinetic mechanism of plasminogen activation by streptokinase

Streptokinase (SK) activates plasminogen (Pg) by specific binding and nonproteolytic expression of the Pg catalytic site, initiating Pg proteolysis to form the fibrinolytic proteinase, plasmin (Pm). The SK-induced conformational activation mechanism was investigated in quantitative kinetic and equilibrium binding studies. Progress curves of Pg activation by SK monitored by chromogenic substrate hydrolysis were parabolic, with initial rates (v(1)) that indicated no transient species and subsequent rate increases (v(2)). The v(1) dependence on SK concentration for [Glu]Pg and [Lys]Pg was hyperbolic with dissociation constants corresponding to those determined in fluorescence-based binding studies for the native Pg species, identifying v(1) as rapid SK binding and conformational activation. Comparison of [Glu]Pg and [Lys]Pg activation showed an approximately 12-fold higher affinity of SK for [Lys]Pg that was lysine-binding site dependent and no such dependence for [Glu]Pg. Stopped-flow kinetics of SK binding to fluorescently labeled Pg demonstrated at least two fast steps in the conformational activation pathway. Characterization of the specificity of the conformationally activated SK.[Lys]Pg* complex for tripeptide-p-nitroanilide substrates demonstrated 5-18- and 10-130-fold reduced specificity (k(cat)/K(m)) compared with SK.Pm and Pm, respectively, with differences in K(m) and k(cat) dependent on the P1 residue. The results support a kinetic mechanism in which SK binding and reversible conformational activation occur in a rapid equilibrium, multistep process.     

Using fluorescence correlation spectroscopy to study conformational changes in denatured proteins

Fluorescence correlation spectroscopy (FCS) is a sensitive analytical tool that allows dynamics and hydrodynamics of biomolecules to be studied under a broad range of experimental conditions. One application of FCS of current interest is the determination of the size of protein molecules in the various states they sample along their folding reaction coordinate, which can be accessed through the measurement of diffusion coefficients. It has been pointed out that the analysis of FCS curves is prone to artifacts that may lead to erroneous size determination. To set the stage for FCS studies of unfolded proteins, we first show that the diffusion coefficients of small molecules as well as proteins can be determined accurately even in the presence of high concentrations of co-solutes that change the solution refractive index significantly. Indeed, it is found that the Stokes-Einstein relation between the measured diffusion coefficient and solution viscosity holds even in highly concentrated glycerol or guanidinium hydrochloride (GuHCl) solutions. These measurements form the basis for an investigation of the structure of the denatured state of two proteins, the small protein L and the larger, three-domain protein adenylate kinase (AK). FCS is found useful for probing expansion in the denatured state beyond the unfolding transition. It is shown that the denatured state of protein L expands as the denaturant concentration increases, in a process akin to the transition from a globule to a coil in polymers. This process continues at least up to 5 M GuHCl. On the other hand, the denatured state of AK does not seem to expand much beyond 2 M GuHCl, a result that is in qualitative accord with single-molecule fluorescence histograms. Because both the unfolding transition and the coil-globule transition of AK occur at a much lower denaturant concentration than those of protein L, a possible correlation between the two phenomena is suggested.     

Using ligand-induced conformational change to screen for compounds targeting G-protein-coupled receptors

The authors describe a novel drug strategy designed as a primary screen to discover either antagonist or agonist compounds targeting G-protein-coupled receptors (GPCRs). The incorporation of a nuclear localization sequence (NLS, a 5 amino acid substitution), in a location in helix 8 of the GPCR structure, resulted in ligand-independent receptor translocation from the cell surface to the nucleus. Blockade of the GPCR-NLS translocation from the cell surface was achieved by either antagonist or agonist treatments, each achieving their result in a sensitive concentration-dependent manner. GPCR-NLS translocation and blockade occurred regardless of the identity of the G-protein-coupling, and thus this assay is also ideally suited for identification of compounds targeting orphan GPCRs. The GPCR-NLS trafficking was visualized by fusion to fluorescent detectable proteins. Quantification of this effect was measured by determining the density of cell surface receptors, using enzyme fragment complementation in a manner suitable for high-throughput screening. Thus, the authors have developed a cellular assay for GPCRs suitable for compound screening without requiring prior identification of an agonist or knowledge of G-protein-coupling.     

FRET detection of cellular alpha4-integrin conformational activation

Integrins are cell adhesion receptors, expressed on every cell type, that have been postulated to undergo conformational changes upon activation. Here, different affinity states were generated by exposing alpha4-integrins to divalent ions or by inside-out activation using a chemokine receptor. We probed the dynamic structural transformation of the integrin on live cells using fluorescence resonance energy transfer (FRET) between a peptide donor, which specifically binds to the alpha4-integrin, and octadecyl rhodamine B acceptors incorporated into the plasma membrane. We analyzed the data using a model that describes FRET between a random distribution of donors and acceptors in an infinite plane. The distance of closest approach was found to vary with the affinity of the integrin. The change in distance of closest approach was approximately 50 A between resting and Mn2+ activated receptors and approximately 25 A after chemokine activation. We used confocal microscopy to probe the lateral organization of donors and acceptors subsequent to integrin activation. Taken together, FRET and confocal results suggest that changes in FRET efficiencies are primarily due to the vertical extension of the integrin. The coordination between the extension of alpha4-integrin and its affinity provides a mechanism for Dembo's catch-bond concept.     

Using a variant of coverslip hypoxia to visualize tumor cell alterations at increasing distances from an oxygen source

Early stages in tumor development involve growth in confined spaces, where oxygen diffusion is limited and metabolic waste products accumulate. This hostile microenvironment imposes strong selective pressures on tumor cells, leading eventually to the survival and expansion of aggressive subclones that condition further tumor evolution. To model features of this microenvironment in vitro, a diffusional barrier can be introduced in the form of a coverslip placed on top of cells, a method termed coverslip hypoxia. Using a variant of this method, with larger volume between coverslip and cells and with oxygen diffusion occurring only through a small hole in the center of the coverslip, we have visualized alterations in LNCaP tumor cells as a function of their distance to the oxygen source at the center. We observed remarkable morphological changes in LNCaP cells as the distance from the center increases, with cells becoming highly spread, displaying dynamic membrane protrusions and occasionally adopting a migratory phenotype. Concomitantly, cells farther from the center displayed marked increases in the hypoxia marker hypoxyprobe, whereas extracellular pH decreased in the same direction. Cells with altered morphology displayed prominent increases in fibrillar actin, as well as swollen mitochondria with distorted cristae and accumulation of neutral lipid-containing intracellular vesicles. These results show that an in vitro microenvironment that models diffusional barriers encountered by tumors in situ can have profound effects on tumor cells. The coverslip hypoxia variant we describe can be used to characterize in vitro the response of tumor cells to environmental conditions that play crucial roles in early tumor development.